Fusion solo chemiluminescence system

Proteins in the samples (20 μg protein/lane) were separated by electrophoresis on a 10% sodium dodecyl sulfate–polyacrylamide gel and further transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with primary antibodies (Cell Signaling, Beverly, MA, USA) against IKKα, phospho-IKKα/β, IKKβ, I-κBα, phospho-I-κBα, NF-κB p65, phospho-NF-κB p65, iNOS, COX-2, and GAPDH for 1 h at room temperature. After binding with HRP-conjugated anti-rabbit antibodies (Cell Signaling, Beverly, MA, USA) for 1 h, the PVDF membranes were developed using enhanced chemiluminescence (ECL) Advance Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK) and visualized using a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Germany).

Western blotting analysis was used to assess expression of target proteins. Briefly, after treatment, the harvested cells were washed twice with cold phosphate-buffered saline, and total cell lysates were prepared with radio immunoprecipitation assay buffer (RIPA buffer, Cell Signaling Technology, Inc., Beverly, MA, USA) supplemented with 1 × EDTA-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions.
Protein content was quantified via bicinchoninic acid (BCA) protein assay, and 20 μg, along with molecular weight markers, was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), for 90 min at 110 V. This was then transferred to a polyvinylidene fluoride (PVDF) transfer membrane and immunoblotted with corresponding antibodies. Immunodetection was performed using the ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, UK) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) [59 (link)].

3T3-L1 cells were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well for 24 h and treated with compound 1 at indicated concentrations (0, 25, 50, 100 µM). After 24 h, cells were collected and lysed in RIPA buffer (Tech&Innovation, Gangwon, Republic of Korea) following the manufacturer’s instructions to obtain whole-cell extracts. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). The proteins separated by electrophoresis were transferred onto PVDF membranes. Primary antibodies, including PPARα, FAS, adiponectin, CPT1A, and β-actin, were used in combination with conjugated secondary antibodies (Cell Signaling, Boston, MA, USA) to label the target proteins. The bound antibodies were detected using Pierce™ ECL Advance Western Blotting Detection Reagents (Thermo Scientific, Waltham, MA, USA) and visualized with the FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

After sample treatments, MCF-7 cells incubated in 6-well plates were collected and lysed with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride on ice. The amounts of each protein were determined using the Pierce™ BCA Protein Assay Kit. Equal amounts of proteins (20 μg/lane) were separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto polyvinylidene difluoride membranes [37 (link)]. After blocking with 5% skim milk for 1 h, the proteins in the membrane were incubated at 25 °C for 1 h with primary antibodies against BID, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, PARP, and GAPDH. Following incubation with HRP-conjugated anti-rabbit secondary antibodies at room temperature for 1 h, the expressed proteins were reacted using ECL Advance Western blotting detection reagents and visualized with a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions.

The LLC-PK1 cells were plated onto 6-well plates at a density of 4 × 10⁵ cells per well. The cells were treated with 25 μM cisplatin for 24 h in the absence or presence of 10 and 25 μM formonetin. Cells were harvested and lysed using a RIPA buffer containing a freshly added protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and further transferred onto a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked with tris-buffered saline containing 5% skim milk and subsequently incubated with primary antibodies against c-Jun N-terminal kinase (JNK), phospho-JNK, JNK, cleaved caspase-8, cleaved caspase-3, Bcl-2, Bax, and GAPDH. The membranes were then incubated with appropriated horseradish peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using ECL Advance Western Blotting Detection Reagents and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) and quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells were seeded at a density of 4 × 10⁵ cells per/well in 6-well plates. One day after seeding, cells were treated for 2 h with the test samples. Then, 25 μM cisplatin was added to wells for 24 h. Untreated cells were used as controls. After incubation, cells were washed twice with cold phosphate-buffered saline and scraped off the plates, and then lysed using RIPA buffer (Cell Signaling Technology, Inc., Beverly, MA, USA) supplemented with 1× EDTA free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions. Aliquots of 20 μg protein were subjected to SDS PAGE (10% gels) along with molecular weight markers for 90 min at 110 V, and then transferred to PVDF transfer membranes. Membranes were probed with primary antibodies to phospho-JNK, JNK, phospho-p38, p38, cleaved caspase-3, GAPDH and then with secondary immunoglobulin G horseradish peroxidase conjugates. Bound antibodies were detected using ECL Advance Western Blotting Detection reagents (GE Healthcare, Cambridge, UK) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).